Mechanosensitive ion channels (MSCs) are membrane-bound proteins that let water and solute molecules flow in and out of the cell in response to membrane deformation (23). They are essential to life by letting the cell respond to osmotic changes (24). Eukaryotic MSCs are involved in sensory modalities, like touch, sound, fluid balance, and blood pressure (15,25). Problems with these channels are implicated in cardiac arrhythmia, muscular dystrophy, glioma, pathological pain, neurovestibular disturbances, and tumour metastasis (13,26- 28). MSCs (of large conductance, MscL, and of small conductance, MscS) from bacteria are best understood. This may aid in attacking bacteria via antibiotics (29) and may help understand the eukaryotic MSCs, which are poorly characterized (30). Yet vital information about the bacterial MSCs and especially eukaryotic MSCs, are missing. For the bacterial channels, the MscL channel has been crystallized in the closed form, but not in the open form (31). The open pore of the MscL has been experimentally tested via several ensemble techniques, including EPR and ensemble FRET, but systematic errors likely result in an overestimation (32), or an underestimation (33,34), or have not been sensitive to the requisite distances (35). We propose to study the open and closed states of MscL using single molecule fluorescence energy transfer (smFRET), a technique I helped to invent (36,37). This will be supplemented with molecular dynamics, led by Klaus Schulten (U. Illinois, Urbana-Champaign) (38,39) who has studied the MscL/MscS channels (1,3,4,6,7). Our other collaborator, Boris Martinac (Victor Chang Institute, Australia) has extensively studied the MscL/MscS (32-35,40-46). We will study how the MscL opens. The leading candidates are the barrel-stave model, with one transmembrane helices (TM1) moving, and the helix-tilt model (47), with two helices (TM1 and TM2) moving. (47). We have formed the MscL channel in both the open and closed state, and find that smFRET indicates that both helices move, leading to a change in the pore diameter of 2.8 nm. We therefore argue strongly for the helix-tilt model. We also propose to study the behavior of the cytoplasmic (CP) helix, a source of significant controversy (48,49). We have preliminary smFRET data, which argues that the CP domain is dissociated. Furthermore, we propose to investigate the interaction between the channel and its surrounding membrane by studying the conformational dynamics of the channel using a mutant (G22N) (49) which spontaneously opens and closes. We also propose to investigate the process of channel opening by independent MD simulations for which we have successfully simulated the opening of the pore by forcing water through the channel. Finally, in collaboration with Philip Gottlieb, SUNY, Buffalo, we have preliminary results on a huge (>1 MD) eukaryotic MSC, called (tetrameric) Piezo1 (12,30,50). We use both SimPull (17) to isolate a single channel, and super-resolution fluorescence techniques, gSHRImP (18,51) and SHREC (19) (derived from my FIONA technique (20)), to study select positions. Results suggest that the N-to-N distance of the tetramer is ~52 nm.